Method and compositions for solubilization of pentacyclic triterpenes

ABSTRACT

The present invention provides compositions and methods for the solubilization of poorly soluble drugs such as triterpenes like betulinic acid and/its derivatives in pharmaceutically acceptable liquid vehicles that avoid use of potentially toxic solvents that are often used for the solubilization of poorly soluble drugs. In the compositions of this invention the drugs remain physically and chemically stable and can be administered intravascularly without undue toxicity from undissolved drug and/or from the solvent vehicles at a drug dose meant to be effective to exhibit clinically significant anticancer activity.

FIELD OF THE INVENTION

[0001] The invention relates to compositions of poorly water-solubledrugs such as triterpenes and methods for preparing the compositions.Particularly, the invention relates to compositions comprisingpentacyclic triterpenes and more particularly, betulinic acid and itsderivatives. The present invention also relates to betulinic acid and/orits derivatives that are used for the treatment and suppression ofmalignant diseases including but not limited to leukemias, lymphomas,melanoma, prostate and ovarian cancers.

BACKGROUND OF THE INVENTION

[0002] Under the auspices of a National Cooperative Natural Product DrugDiscovery Group supported by the National Cancer Institute, thepotential anti-tumor activity of approximately 2500 extracts derivedfrom globally collected plants were evaluated in a panel of enzyme basedassays and in a battery of cultured human tumor cell lines. One suchextract, prepared from the stem bark of Ziziphus mauritiana Lam.(Rhamnaceae) displayed selective cytotoxicity against cultured humanmelanoma cells (Nature Medicine, Vol. 1 (10) 1995, WO 96/29068). As aresult of bioactivity guided fractionation, betulinic acid, apentacyclic triterpene, was identified as a melanoma-specific cytotoxicagent. In follow-up studies conducted with athymic mice carrying humanmelanomas, tumor growth was completely inhibited without toxicity. Asjudged by a variety of cellular responses, anti-tumor activity wasmediated by the induction of apoptosis.

[0003] A number of triterpenoids, including betulinic acid, have severalknown medical applications, including use as an anticancer drug.Anderson et al., in WO 95/04526, discuss derivatives of triterpenoidswhich have been used in cancer therapy, including their activity againstpolyamines which are required by cells to grow at an optimal rate. Someof these triterpenoids have been found to interfere with enzymaticsynthesis of polyamines required for optimal cell growth, and thusinhibit the growth of cancer cells, particularly by inhibiting ornithinedecarboxylase (Yasukawa, K. et al. Oncology 48:72-76, 1991). Theanti-cancer activity of betulinic acid and some derivatives has beendemonstrated using mouse sarcoma 180 cells implanted subcutaneously innude mice (JP 87,301,580). Choi et al have shown that betulinic acid3-monoacetate, and betulinic acid methyl ester exhibit ED50 values of10.5 and 6.8 μg/ml, respectively against P388 lymphocytic leukemia cells(Choi, Y. H. et al. Planta Medica vol. XLVII, pages 511-513, 1988).Betulinic acid and derivatives of betulinic acid have highly selectiveactivity against melanoma cells, murine carcinosarcoma and murinelymphocytic leukemia.

[0004] The selective cytotoxicity of betulinic acid and its variousderivatives, and their lack of toxicity towards normal cells, afford afavorable therapeutic index. However, the poor solubility of betulinicacid and its derivatives has limited research on other activities ofbetulinic acid and its derivatives. This is also reflected by the factthat except for a few topical preparations no systemic preparations forthe administration of betulinic acid and/or its derivatives arereported. This is due to very poor aqueous solubility of the betulinicacid and its derivatives. To date there is no formulation reported forthe administration of these drugs for human use.

[0005] There is a need, therefore, for solubilization of betulinic acidand/or its derivatives into pharmaceutically acceptable compositionswhich are miscible with aqueous intravenous diluting fluids.

[0006] It is an important objective of the present invention tosolubilize derivatives of betulinic acid and/or its derivatives thathave limited utility due to poor solubility.

[0007] It is another important objective of the present invention toovercome the poor solubility of the betulinic acid and its derivativesby solubilizing them in a pharmaceutically acceptable non-toxic solventsystem.

[0008] Another very important objective of the present invention is toprovide a pharmaceutically acceptable composition of betulinic acid andits derivatives that can be utilized for the treatment of malignantdiseases.

SUMMARY OF THE INVENTION

[0009] The present invention aims to overcome the above problems andrealize the objects of the present invention by providing a novel,pharmaceutically acceptable solvent system based, inter alia, upon theprinciples of co-solvency. The solvent system comprises an organicsolvent, a co-solvent and a solubilizer.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides compositions and methods for thesolubilization of poorly soluble drugs such as triterpenes likebetulinic acid and/its derivatives in pharmaceutically acceptable liquidvehicles that avoid use of potentially toxic solvents that are oftenused for the solubilization of poorly soluble drugs. In the compositionsof this invention the drugs remain physically and chemically stable andcan be administered intravascularly without undue toxicity fromundissolved drug and/or from the solvent vehicles at a drug dose meantto be effective to exhibit clinically significant anticancer activity.Before intravascular administration, the compositions are diluted in apharmaceutically acceptable solution that is suitable for intravascularadministration.

[0011] According to the present invention, a triterpene is dissolved inan organic solvent, followed by the addition of a co-solvent and asolubilizer. Examples of organic solvents that can be used aredimethylacetamide (DMA), dimethylsulphoxide (DMSO), and alcohols such asmethanol, ethanol, propanol and isopropanol. Examples of co-solventsthat can be used are polyethylene glycol (PEG), ethanol, various amidessuch as pyrrolidinone or 1-methyl-2-pyrrolidinone, or sulfur containingcompounds such as sulfolane, dimethylsulfoxide (DMSO) or tetramethylenesulfoxide. Any other co-solvent that would be suitable for use in thisinvention can also be used. Preferably, polyethylene glycol (PEG) isused as the co-solvent. The molecular weight of the PEG may vary fromabout 300 to about 10,000. More preferably the PEG has an averagemolecular weight of about 300. Examples of solubilizers that can be usedinclude polyoxyethylene sorbitan fatty acid esters, poloxamers,polyoxyethylene stearates or lecithin. A polyoxyethylene sorbitan fattyacid ester such as POLYSORBATE 20, 21, 40, 60, 61, 65, 80, 81, 85 or120, or TWEEN 80, preferably POLYSORBATE 80 or TWEEN 80, a poloxamersuch as a PLURONIC or a polyoxyethylene stearate such as MYRJ 52 can beused as the solubilizer. Any other solubilizer that would be suitablefor use in this invention can be used.

[0012] According to the invention, betulinic acid or any of itsderivatives is dissolved in an organic solvent as the primary vehicle,for example, dimethylacetamide (DMA), followed by the addition ofco-solvent(s) such as polyethylene glycol and a solubizer such aspolyoxyethylene sorbitan fatty acid ester to retain the drug in solutionupon dilution with an aqueous vehicle.

[0013] The preferred compositions utilize combinations of anhydrousdimethylacetamide (DMA), as the primary solvent, polyethylene glycol 300(PEG) as the co-solvent and a polyoxyethylene sorbitan fatty acid estersuch as POLYSORBATE 80 as the solubilizer. Such compositions aremiscible in solutions that are used in intravenous preparations. Theseintravenous solutions include but are not limited to solutions thatcontain 2-25% by weight of dextrose such as 5% dextrose solution, 10%dextrose solution, normal saline or dextrose-normal saline. Anyintravenous solution that can be used for humans or animals can be used.These intravenous solutions are examples of vehicles in which betulinicacid or its derivatives are effectively solubilized and can beadministered to humans or animals, alone or in combination with otherdrugs.

[0014] Accordingly, the present invention provides a novelpharmaceutical composition for solubilization of triterpenes whichcomprises from 5% to 50% by volume of an organic solvent, from 20% to80% by volume of a co-solvent and from 5% to 60% by weight of asolubilizer. Optionally, the composition is diluted with an aqueousintravenous diluting fluid.

[0015] In one embodiment of the invention the composition forsolubilization of triterpenes comprises from 5-50% of dimethylacetamide(DMA), from 20-80% by volume of polyethylene glycol (PEG) and from 5-60%by weight of polyoxyethylene sorbitan fatty acid ester such as Tween.

[0016] In a preferred embodiment, the pharmaceutical composition forsolubilization of triterpenes comprises from 20% to 50% by volume ofdimethylacetamide (DMA), from 20% to 40% by volume of polyethyleneglycol (PEG) and from 5% to 30% by weight of polyoxyethylene sorbitanfatty acid ester such as Tween. Optionally, the composition is dilutedwith an aqueous intravenous diluting fluid.

[0017] In a still more preferred embodiment, the DMA and polyoxyethylenesorbitan fatty acid ester such as TWEEN 80 are present at a ratio ofabout 1:2 (v/v) and in another preferred embodiment, the DMA and PEG 300are present at a ratio of about 1:4(v/v).

[0018] It will be understood by a skilled artisan that the presentinvention will work even outside these ratios.

[0019] Preferably the triterpene is betulinic acid

[0020] or a derivative thereof.

[0021] Given below is the list of some of the representative derivativesof betulinic acid that can be solubilized using the above compositions.

[0022] Wherein, Betulinic acid derivatives representing R, R₁, R₂, R₃,and R₄ are selected from the following as shown in the Table hereinbelow:

Derivatives R R₁ R₂ R₃ R₄ MJ-321-RS H H —OCOCH₃ H CH₂═CCH₃ MJ-351-RS H H═NOH H CH₂═CCH₃ MJ-353-RS H H ═NNHC₆H₅ H CH₂═CCH₃ MJ-408-RS H H —OCOCH₃CH₂COOCH₃ CH₂═CCH₃ MJ-481-RS H H ═NOCOCH₃ H CH₂═CCH₃ MJ-542-RS H Br ═O H—CH(CH₃)₂ MJ-606-RS H H —OCOC₆H₅ H —CH(CH₃)₂ MJ-807-RS H H ═NNHCOC₆H₅ H—CH(CH₃)₂ MJ-912-RS H H —NH NH C₆H₄OCH₃(4) H —CH(CH₃)₂ MJ-939-RS H H —OCO C₆H₄CF₃(3) H CH₂═C—CH₃ MJ-1025-RS H H —NHCOCH₃ —CH₂COOCH₃ —CH(CH₃)₂MJ-1101-RS H H —OH —COCH═CH₂ CH₂═C—CH₃ MJ-1207-RS H H ═NNHC₆H₃Cl₂(3,4) HCH₂═C—CH₃ MJ-1305-RS H H —OCOC₆H₄Cl(4) H —CH(CH₃)₂ MJ-1324-RS H H═NOC₂H₅ H —CH(CH₃)₂ MJ-1396-RS H H —N[COC₆H₃F₂(2,4)]OCH₂C₆H₄NO₂(4) H—CH(CH₃)₂ MJ-1402-RS H H —O Morpholinoyl H —CH(CH₃)₂ MJ-1456-RS H H—NHOCH₂C₆H₄[NHCH₂C₆H₃F₂(3,4)](4) H —CH(CH₃)₂ MJ-1464-RS H Br ═NNHCOC₆H₅H —CH(CH₃)₂ MJ-1498-RS H H —NHOCH₂C₆H₄(NHSO₂CH₃)(4) Y —CH(CH₃)₂MJ-1529-RS H Br —OCOCH₃ Y —CH(CH₃)₂ MJ-1103-RS H H —OH —COCH═CH₂—CH(CH₃)₂ MJ-1104-RS H H —OCOC₆H₄(C₅H₁₁)(4) H CH₂═CCH₃ MJ-1602-RS H Br—OH Y —CH(CH₃)₂ MJ-1623-RS H H —OH —COOH —COCH₃ MJ-548-RS H Br ═OCH₂CH₂COOCH₃ —CH(CH₃)₂ MJ-937-RS H H —OCOC₆H₃F₂(2,4) H —CH(CH₃)₂MJ-998-RS H H —N═CHC₆H₃F₂(3,4) H —CH(CH₃)₂ MJ-1065-RS H H—N═CHC₆H₃F₂(2,4) H —CH(CH₃)₂ MJ-1098-RS H H ═NOCH₂C₆H₄NO₂(4) H —CH(CH₃)₂MJ-476-RS H H ═O —COOCH₂COOCH₃ —CH(CH₃)₂ MJ-568-RS H H OH —COOH—CH(CH₃)₂ MJ-456RS H H ═O —COOCH₂COOCH₃ —CH₂═C—CH₃

[0023] In a preferred embodiment the concentration of triterpene isabout 0.001 to 40 mg/ml of the composition before it is diluted with anintravenous solution.

[0024] The present invention also provides a method for solubilizingtriterpenes which comprises the steps of:

[0025] dissolving said triterpene in from 5% to 50% by volume of anorganic solvent, from 20% to 80% by volume of a co-solvent and from 5%to 60% by weight of a solubilizer and if desired, diluting saidcomposition with an aqueous intravenous diluting fluid.

[0026] The present invention also provides a method for solubilizingtriterpenes which comprises the steps of:

[0027] dissolving said triterpene in from 5% to 50% by volume ofdimethylacetamide (DMA), from 20% to 80% by volume of polyethyleneglycol (PEG) and from 5% to 60% by weight of polyoxyethylene sorbitanfatty acid ester and if desired, diluting said composition with anaqueous intravenous diluting fluid.

[0028] Preferably, said triterpenes are selected from betulinic acid andits derivatives. More preferably, PEG is added to the solution ofbetulinic acid or its derivatives at a ratio of DMA:PEG of about 1:4(v/v), followed by the addition of a polyoxyethylene sorbitan fatty acidester such as TWEEN 80 at a ratio of DMA: polyoxyethylene sorbitan fattyacid ester of about 1:2 (v/v) to obtain a concentration of betulinicacid or its derivatives of about 0.001 mg/ml to 40 mg/ml. The solutioncan be diluted to a concentration of triterpene between 0.0001 to lessthan 40 mg/ml.

[0029] The present invention will now be described with reference to thefollowing non-limiting examples.

EXAMPLE 1

[0030] Enhanced solubility of betulinic acid in physiologicallyacceptable solvents.

[0031] The solubility of betulinic acid and its derivatives wasdetermined in various individual solvents. Based on the solubility ofbetulinic acid in these particular vehicles, it was then attempted toenhance the solubility of betulinic acid by mixing different solventsaccording to the principles of co-solvency (Spiegel and Noseworthy,1963, Yalkowsky and Roseman, 1981). Different solvent systems wereevaluated to arrive at a suitable relevant stock composition. The stockcomposition would then be diluted with a final solvent or intravenoussolution to yield a working formulation of 0.001 -40mg/ml.

EXAMPLE 2

[0032] Several methods and compositions were evaluated for their abilityto solubilize betulinic acid and its derivatives in water miscible,physiologically acceptable vehicles. The solvents that were triedincluded DMA, dimethylsulfoxide (DMSO), absolute ethanol, glycerin,polyethylene glycols (PEGs) and propylene glycol, in addition to theaqueous diluting fluids normal saline, and 2-25% dextrose in solution.The solubility's in these solvents is shown in the following table.SOLVENT SOLUBILITY mg/ml N,N Dimethylacetamide (DMA) ≧200 mg/mlDimethylsulfoxide (DMSO) ≦100 mg/ml Polyethylene glycols <5 mg/ml (MW200, 300, 400) Absolute ethanol varies between 3-10 mg/ml depending uponthe derivative Glycerin insoluble Propylene glycol <5 mg/ml Waterinsoluble

[0033] DMA and DMSO were found to be the best solvents, whereasbetulinic acid and its derivatives were insoluble in most of the aqueoussolvents. Although DMSO has been used as a solvent for parenteralformulations of anti-cancer agents, it is not a preferred vehicle fordissolving drugs due to its toxicity and potential incompatibilitieswith plastic administration devices. Therefore DMA was chosen as thesolvent of choice for dissolving the drug. DMA, per se, could dissolve aconcentration of betulinic acid and its derivatives up to 250 mg/ml.However, a concentrated solution of betulinic acid or its derivatives inDMA alone cannot be administered as such in the body as the total volumeof the solvent administered in the body would exceed the toxicity limit.Moreover, the solution on dilution with aqueous fluid in the body maylead to precipitation of drug from the solution leading to increaseddrug toxicity.

[0034] The following sets of studies were performed to arrive atphysiologically acceptable formulations for intravenous administration.

EXAMPLE 3

[0035] Solubilizing of betulinic acid and/or its derivatives using DMAand PEG.

[0036] A concentrated solution of betulinic acid or its derivatives wasprepared by dissolving 50-200 mg of betulinic acid or its derivatives in1 ml of DMA. The above stock solution was diluted with PEG (polyethyleneglycol, MW 300) in a ratio of 1:4 to a concentration of 10-40 mg/ml.Betulinic acid/derivatives 200 mg DMA 20% v/v PEG 300 80% v/v

[0037] However, on dilution with the aqueous diluting fluid, the drugimmediately precipitated out. Therefore, in further trials the drugconcentration was reduced. Betulinic acid/derivatives 50 mg DMA 20% v/vPEG 300 80% v/v

[0038] The above solution also on dilution with the aqueous dilutingfluid, led to immediate precipitation of drug even at concentrations aslow as 0.001-0.1 mg/ml.

[0039] Therefore, in further trials an attempt was made to increase theproportion of DMA to enhance the solubility of the drug on dilution.

EXAMPLE 4

[0040] Betulinic acid/derivatives 50 mg DMA 50% v/v PEG 300 50% v/v

[0041] The 1:1 ratio of DMA and PEG improved the solubility of betulinicacid or its derivatives on dilution with aqueous diluting fluid like2-25% dextrose, or normal saline. However, 1:1 ratio of DMA and PEG inthe presence of betulinic acid or its derivatives and on dilution withaqueous diluting fluid gave a viscous solution which gels over timealthough there are no signs of any precipitation of drug.

EXAMPLE 5

[0042] To further improve the solubility of betulinic acid and itsderivatives on dilution with aqueous diluting fluids, additions ofsurfactants were investigated. The surface-active (solubilizers) agentsmost commonly used in the prior art for enhancing the solubility ofpoorly soluble drugs are polyoxyethylene sorbitan monooloeate(Polysorbate 80) and polyoxyethylene polyoxypropylene ethers (Pluronic68). Less commonly used surface active agents (solubilizers) inparenteral products are lecithins, Polyoxyl 40 stearate (MYRJ 52).

[0043] In the following examples one or more co-solvents with asolubilizers or a combination of (solubilizers) was added to keep thedrug in solution after diluting with an aqueous intravenous dilutingfluid.

EXAMPLE 6

[0044] Betulinic acid/derivatives 10-20 mg DMA 50% v/v Tween 80 50% w/v

[0045] The above combination could dissolve upto 20 mg/ml of the drug.However, the drug immediately precipitated out on dilution with aqueousdiluting fluid.

EXAMPLE 7

[0046] Betulinic acid/derivatives 10-20 mg Absolute ethanol 20-40% v/vTween 80 60-80% w/v

[0047] The above composition could dissolve upto 20 mg/ml of the drug.However, the drug solution only remained clear for a maximum of 3 hourson dilution with aqueous diluting fluid to a concentration of 0.3-1.4mg/ml.

EXAMPLE 8

[0048] Betulinic acid/derivatives 60 mg Absolute ethanol 65% v/v Tween80 5% w/v PEG 300 30% v/v

[0049] The above composition could dissolve up to 6 mg/ml of the drug.The drug solution remained clear on dilution with aqueous diluting fluidto a concentration of 0.08-0.4 mg/ml.

EXAMPLE 9

[0050] In this experiment, the amount of the organic solvent wasreduced. Betulinic acid/derivatives 40 mg Absolute ethanol 35% v/v Tween80 15% w/v PEG 300 50% v/v.

[0051] The above composition could dissolve only up to 4 mg/ml of thedrug. The drug solution remained clear on dilution with aqueous dilutingfluid to a concentration of 0.08 to 0.4 mg/ml. However, outside thisrange there was immediate precipitation.

EXAMPLE 10

[0052] Betulinic acid/derivatives 30 mg DMA 10% v/v Propylene glycol 20%v/v Absolute ethanol 35% v/v Tween 80 35% w/v

EXAMPLE 11

[0053] Betulinic acid/derivatives 150 mg DMA 20% v/v Propylene glycol10% v/v Absolute ethanol 30% v/v Tween 80 40% w/v

[0054] Betulinic acid and almost all of its derivatives were found to besoluble in the above system in a concentration range of 2-15 mg/ml whichon dilution with an aqueous diluting fluid remains clear at a lowerconcentration of 0.012-2 mg/ml. However, these compositions had a quitehigh amount of the organic solvents which is not desirable as these mayprove toxic on administration in the body. Therefore, attempts wereundertaken to solubize the maximum quantity of the drug in a minimumamount of the organic solvents with a less toxic co-solvent andsurfactant combination.

EXAMPLE 12

[0055] Betulinic acid/derivatives 5-20 mg/ml DMA 20% v/v MYRJ 52 30% w/vTween 50% w/v

EXAMPLE 13

[0056] Betulinic acid/derivatives 5-20 mg/ml DMA 20% v/v MYRJ 52 20% w/vTween 80 20% w/v PEG 300 40% v/v

[0057] The above two compositions containing MYRJ 52 could dissolve upto20 mg/ml of the drug but on dilution with aqueous diluting fluid to aconcentration to 0. 1-1.0 mg/ml, the drug precipitated out in a fewhours. Moreover, preparations containing MYRJ 52 tend to solidify atlower temperatures. Also MYRJ 52 is not a preferred solubilizing agentfor parenteral products because of its higher toxicity. Therefore it wasattempted to replace MYRJ 52 with Pluronic 68 and lecithin, which arecomparatively safer for intravenous administration.

EXAMPLE 14

[0058] Betulinic acid/derivatives 2-15 mg/ml DMA 20% v/v Tween 80 20%w/v PEG 300 57% v/v Poloxamer 407  3% w/v

EXAMPLE 15

[0059] Betulinic acid/derivatives 2-15 mg/ml DMA 20% v/v Tween 80 20%w/v Lecithin  5% w/v PEG 300 55% v/v

[0060] The above compositions could dissolve 2-15 mg/ml of betulinicacid and its derivatives. However, on dilution with the aqueous dilutingfluid the drug remained soluble only at lower dilutions (0.001-1 mg/ml).

EXAMPLE 16

[0061] After conducting a number of trials as described above tosolubilize betulinic acid and its derivatives the most preferredcomposition was the one containing DMA, PEG 300 and Tween 80. The threecomponents were tried in varying proportions as mentioned in thefollowing example. Up to 22 mg/ml of betulinic acid and/or itsderivatives could be dissolved in these compositions. On dilution withan aqueous diluting fluid to a concentration of 0.1-10 mg/ml thecomposition remained clear for more than 5 days without anyprecipitation of the drug. Betulinic acid/derivative 10-20 mg/ml DMA20-50% v/v PEG 300 20-40% v/v Tween 80 5-30% w/v

1. A composition comprising a triterpene, from 5% to 50% by volume of anorganic solvent, from 20% to 80% by volume of a co-solvent and from 10%to 60% by weight of a solubilizer.
 2. A composition comprising atriterpene, from 5% to 50% by volume of dimethylacetamide, from 20% to80% by volume of polyethylene glycol and from 10% to 60% by weight ofpolyoxyethylene sorbitan fatty acid ester.
 3. The composition accordingto claim 1, wherein the triterpene is betulinic acid or a derivativethereof.
 4. The composition according to claim 2, wherein the triterpeneis betulinic acid or a derivative thereof.
 5. The composition accordingto claim 4, wherein the triterpene is at a concentration of about 0.001to 40 mg/ml.
 6. The composition according to claim 1, wherein theorganic solvent is selected from dimethylacetamide, dimethylsulphoxideor alcohols.
 7. The composition according to claim 1, wherein theco-solvent is selected from polyethylene glycol, ethanol, an amide,sulfolane, dimethylsulfoxide (DMSO) or tetramethylene sulfoxide.
 8. Thecomposition according to claim 7, wherein the polyethylene glycol has amolecular weight of between 300-10,000.
 9. The composition according toclaim 1, wherein the solubilizer is selected from polyoxyethylenesorbitan fatty acid ester, poloxamer, polyoxyethylene stearates orlecithin.
 10. The composition according to claim 1, further comprisingan aqueous diluting fluid.
 11. The composition according to claim 2,further comprising an aqueous diluting fluid.
 12. The compositionaccording to claim 11, wherein the aqueous diluting fluid is selectedfrom 5% dextrose solution, 10% dextrose solution, normal saline, ordextrose-normal saline.
 13. The composition according to claim 2,wherein the dimethylacetamide and the polyethylene glycol are present ata ratio of 1:4 (v/v).
 14. The composition according to claim 2, whereinthe dimethylacetamide and the polyoxyethylene sorbitan fatty acid esterare present at a ratio of 1:2 (v/v).
 15. A method for preparingcompositions for solubilization of a triterpene which comprises:dissolving the triterpene in from 5% to 50% by volume of a solvent, from20% to 80% by volume of a co-solvent and 5% to 60% by weight of asolubilizer.
 16. The method according to claim 15, further comprisingadding a diluting fluid.
 17. The method according to claim 15, whereinthe solvent is dimethylacetamide, the co-solvent is polyethylene glycoland the solubilizer is polyoxyethylene sorbitan fatty acid ester. 18.The method according to claim 17, further comprising adding a dilutingfluid.